Over two decades ago, it was discovered (Ritossa, F. (1962) Experientia 18:571-573) that specific puffing patterns in the polytene chromosomes of Drosophila busckii could be induced by a brief heat shock. The puffing positions of Drosophila species polytene chromosomes are positions where there is active synthesis of mRNA, thus indicating active gene loci (Beerman, W. (1956) Cold Spring Harbor Symp. Quant. Biol. 21:217-232). Since then it has been shown that a variety of agents, e.g., arsenite or anaerobic conditions, can induce responses similar to those induced by heat, suggesting that a more appropriate name for these genes should be "stress genes". However, the nomenclature of "heat shock" genes is now well established and will be used in the remainder of this application.
Since the early 1950's it was known that the pattern of puffs in the polytene chromosomes of Dipteran larvae changed in a regular manner during development, and it was shown that these changes were controlled by ecdysteroid hormones (Clever, U. and P. Karlson (1960) Exp. Cell. Res. 20:623-626; Becker, H.-J. (1962) Chromosoma 13:341-384). In particular, it was shown that the pattern of puffing was disrupted by a brief heat shock (or treatment with certain chemicals) and resulted in the appearance of three new puffs. The induction of these new puffs was very rapid and occurred within minutes of the heat shock treatment, but the induction was transient. For example, when the temperature was raised from 25.degree..fwdarw.37.degree. C., the puffs reached maximum size within 30 minutes and then regressed. At the same time, all puffs active before the heat shock regressed after the treatment. The heat shock puffing response was also found to occur in all tissues studied and at all stages of development.
It was later found that the heat shock treatment induced synthesis of a small number of polypeptides and repressed the synthesis of most others (Tissieres, A. et al. (1974) j. Mol. Biol. 84:389-398) and the mRNA produced at the new heat shock induced puffs was shown to code for the newly induced polypeptides (Lewis, M. J. et al. (1975) Proc. Nat. Acad. Sci. USA 7.2:3604-3608).
Within a few minutes of heat shock, all polyribosomes break down and are quickly replaced by a new polyribosome peak which contains heat-shock protein mRNA. This mRNA has been hybridized back to the heat shock induced puffs and has been translated in vitro into heat shock proteins (McKenzie, S. et al. (1977) J. Mol. Biol. 117:279-283; Mirault, M. E. et al. (1978) Cold Spring Harb. Syrup. Quant. Biol. 42:819-827). It is of interest to note that even though all polyribosomes break down and the newly induced hs-mRNA's are selectively translated, most normal mRNA's persist during heat shock (Ashburner, N. and J. F. Bonner (1979) Cell 17:241 ff.)
Most of the early work on heat shock genes was done with Drosophila species. However, in 1978, analogous stress responses were found in chick embryonic fibroblasts (Kelly, P. and M. J. Schlesinger (1978) Cell 15:1277-1286), in Chinese hamster ovary cells (Bouche, G. et al. (1979) Nucleic Acids Research 7:1739-1747), in Escherichia coli (Lemeaux, P. G. et al. (1978) Cell 13:427-434), in yeast (Miller, M. J. et al. (1979) Proc. Nat. Acad. Sci. USA 76:5222-5225), in Naegleria (Walsh, C. (1980) J. Biol. Chem. 225:2629-2632), in Tetrahymena (Fink, K. and E. Zeutheu (1978) ICN-UCLA Symp. Mol. Cell. Biol. 12:103-115) and in many other species, Including plants (Barnett, T. et al. (1980) Dev. Genet. 1:331-340). A similar pattern of heat shock protein synthesis has also been reported for tobacco and soybean cells growing in solution culture (Barnett, T., et al. (1980) supra) as that reported for soybean seedling tissue. It was also shown that the effects of trauma on vertebrate cells was similar to the effects of heat shock (Hightower, L. E. and F. P. White (1981) J. Cell. Physiol. 108:261).
The transcriptional and translational control of heat shock genes may be autoregulatory. Thus the activity of these genes may be controlled by the concentrations of the heat shock proteins present in the cells. Therefore, inducers of heat shock genes would be factors that either destroyed the heat shock proteins or rendered them to be effectively unavailable within the cell, e.g., by binding to various cell organelles.
The activation and subsequent repression of heat shock genes in Drosophila has been studied by the introduction of cloned segments into Drosophila cells. The P-element-mediated transformation system, which permits introduction of cloned Drosophila genes into the Drosophila germline, was used (Rubin, G. M. and A. C. Spradling (1982) Science 218:348-353). A gene integrated in this way is often present as a stable, single copy and has a relatively constant activity at a variety of chromosomal locations (Scholnick, S. B. et al. (1983) Cell 34:37-45; Goldberg, D. et al. (1983) Cell 34:59-73; Spradling, A. C. and G. M. Rubin (1983) Cell 34:47-57). In particular the Drosophila hsp70 gene was fused in phase to the E. coli .beta.-gal actosidase structural gene, thus allowing the activity of the hybrid gene to be distinguished from the five resident hsp70 heat shock genes in the recipient Drosophila. Drosophila heat shock genes have also been introduced and their activity studied in a variety of heterologous systems, and, in particular, in monkey COS cells (Pelham, H. R. B. ( 1982) Cell 30:517-528; Mirault, M.-E. et al. (1982) EMBO J. 1:1279-1285) and mouse cells (Corces, V. et al. (1981) Proc. Nat. Acad. Sci. 78:7038-7042).
The hybrid hsp70-lacZ gene appeared to be under normal heat shock regulation when integrated into the Drosophila germ line (Lis, J. T. et al. (1983) Cell 35:403-410). Three different sites of integration formed large puffs in response to heat shock. The kinetics of puff formation and regression were exactly the same as those of the 87C locus, the site from which the integrated copy of the hsp70 gene was isolated. The insertion of the 7 kilobase E. coli .beta.-galactosidase DNA fragment into the middle of the hsp70 structural gene appeared to have had no adverse effect on the puffing response. The .beta.-galactosidase activity in the transformants was regulated by heat shock.
Deletion analysis of the Drosophila hsp70 heat shock promoter has identified a sequence upstream from the TATA box which is required for heat shock induction. This sequence contains hornology to the analagous sequence in other heat shock genes and a consensus sequence CTxGAAxxTTCxAG has been constructed (Pelham, H. R. B. and M. Bienz (1982) EMBO J. 1:1473-1477). When synthetic oligonucl eotides, whose sequence was based on that of the consensus sequence, were constructed and placed upstream of the TATA box of the herpes virus thymidine kinase gene (tk) (in place of the normal upstream promoter element), then the resultant recombinant genes were heat-inducible both in monkey COS cells and in Xenopus oocytes. The tk itself is not heat inducible and probably no evolutionary pressure has occurred to make it heat inducible But the facts above indicate that tk can be induced by a heat shock simply by replacing the normal upstream promoter element with a short synthetic sequence which has homology to a heat shock gene promoter.
An inverted repeat sequence upstream of the TATA box is a common feature of many of the heat shock promoters which have been studied (Holmgren, R. et al. (1981) Proc. Nat. Acad. Sci. USA 78:3775-3778). In five of the seven Drosophila promoters, this inverted repeat is centered at the 5'-side of the penultimate A residue of the consensus sequence, but the sequence of the inverted repeat itself is not conserved (Pelham, H. R. B. (1982) Cell 30:517-528). In some cases, however, the inverted repeat sequence occurs upstream from the TATA box and the consensus sequence is not present. In these cases there is no heat inducibility so the presence of the inverted repeat does not substitute for the consensus sequence.
The functional significance of the heat shock response is not known. Presumably it functions to protect the cell against the environmental stress and to allow the cell to continue its function after the stress situation has passed. These conclusions are supported by a phenomenon known as "acquired thermotolerance". Cells exposed to a single heat shock, or some other stress, are relatively protected against the effects of a second, otherwise lethal heat shock (Li, G. C. and G. M. Hahn (1978) Nature 274:699-701; Henle, K. J. and L. A. Dethlefsen (1978) Cancer Res. 38:1843-1851; Mitchell, H. K. et al. (1979) Dev. Genet. 1:181-192; McAlister, L. and D. B. Finkelstein (1980) Biochem. Biophys. Res. Commun. 93:819-824).
In higher plants, the heat shock (hs) phenomenon was first discovered at the level of protein synthesis in soybeans (Key, J. L. et al. (1981) Proc. Nat. Acad. Sci. USA 78:3526-3530; Barnett, T. et al. (1980) supra). A number of other plants, e.g., pea, millet, corn, sunflower, cotton and wheat, respond similarly to soybean in that a large number of new proteins of similar molecular weight are induced by a heat shock treatment. The major differences that occur among species are the optimum temperature of induction of hs-proteins, the breakpoint temperature (i.e., above this temperature is lethal), the distribution of the 15-20 kD heat shock proteins on two-dimensional gels and the relative level of normal protein synthesis that occurs during heat shock. It has been shown that an elevation of temperature from 28.degree. C. to 40.degree. C. induced de novo synthesis of several major groups of hs-proteins (hsp) whose molecular weights resemble those found for Drosophila. However, there is a marked difference in the complexity of the low molecular weight (lmw) group of hsp's between these two organisms. Drosophila synthesizes four hsp' s of 22, 23, 26 and 27 kilodaltons; soybean produces more than 20 hsp's in the molecular weight range of 15-18 kilodaltons.
The translational preference for hs-mRNA's, while marked, appeared less pronounced in the soybean system (Key, J. L. et al. (1981) supra) than in Drosophila (Storti, R. V. et al. (1980) Cell 22:825-834). The induction of a new set of hs-specific mRNA's in soybean was suggested by in vitro translation of poly(A).sup.+ RNA. Additional evidence for the existence of novel RNA in heat stressed plants was provided by sucrose gradient analysis which showed the accumulation of a 0.49.times.10.sup.6 dalton RNA during hs of tobacco and cowpea leaves (Dawson, W. O. and G. L. Grantham (1981) Biochem. Biophys. Res. Commun. 100:23-30). In Drosophila, where transcriptional control of hs protein synthesis is evident, attempts have been made to find signal structures for coordinate expression of these genes (Holmgren, R. et al. (1981) Proc. Nat. Acad. Sci. USA 78:3775-3778). The influence of hs on poly(A).sup.+ mRNA's of soybean has been assessed using cDNA/poly(A).sup.+ RNA hybridization and cloned cDNA/northern blot hybridization analyses (Schoffl, F. and j. L. Key (1982) J. Mol. Appl. Genet. 1:301-314). The hs response in soybean is characterized by the appearance of a new highly abundant class of poly(A).sup.+ RNA's consisting of some twenty different sequences of an average length of 800 to 900 nucleotides and a decrease in total poly(A).sup.+ RNA complexity associated with changes in relative abundance of the 28.degree. C. sequences. The poly(A).sup.+ RNA's of this new abundant cl ass are present at some 15,000 to 20,000 copies per cell after 2 hours of hs at 40.degree. C. The genes for these four Drosophila hsp's comprise a small hs-gene family with similar sequences which are also related to that of .alpha.-crystallin (Ingolia, T. D. and E. A. Craig (1982) Proc. Nat. Acad. Sci. USA 79:2360-2364) implying that certain structural domains (possibly for functional aggregation) are shared by these proteins. The lmw-hsp genes in soybeans are the most actively expressed and coordinately regulated genes under hs conditions (Schoffl, F. and J. L. Key (1982) J. Mol. Appl. Genet. 1:301-314). Their hsp's are commonly associated with purified nuclei at high temperature, however, and disaggregate at low temperature (Key, J. L. et al. (1982) In: Schlesinger, M. J., Ashburner, M. and A. Tissieres (eds.) Heat shock, from Bacteria to Man. Cold Spring Harbor Laboratory, pp. 329-336). This indicates a common function for these proteins in hs-response which is possibly related to common structural features in proteins and genes. The lmw-hsp genes are subdivided into eight classes defined by sequence homologies among poly(A).sup.+ mRNA's. Two of the eight classes are particularly interesting with respect to gene expression, because they represent the extreme components of the lmw-hsp genes. These are designated classes I and II; I consists of 13 closely related hsp's genes, while II comprises only lhsp which has no known sequence homology to other hs-genes. Later information showed that class II could be grouped with class I. The separation into the two classes was originally made on the basis of a probe distal to the 3'-translated end of pE2019.
A wide range of crop plants respond to elevated temperatures of heat shock conditions by synthesizing a large number (30 or more) of hs-proteins (Key, J. L. et al. (1983) Current Topics in Plant Biochemistry and Physiology, eds. D. D. Randall, D. G. Blevins, R. L. Larson and B. J. Rapp. Vol. 2, Univ. of Missouri, Columbia, pp. 107-117). The high molecular weight hs-proteins were electrophoretically similar among the species. The more complex pattern of low molecular weight (15-27 kd) hs-proteins showed much more electrophoretic heterogeneity between species. Certainly a given soybean hs-cDNA clone showed greater cross hybridization to different soybean hs-poly(A) RNA's than to any hs-RNA from other species, and this limited hybridization with other species was consistent with the observed electrophoretic heterogeneity of the low molecular weight hs-proteins.
The evolutionary conservation of the hs-response across the spectrum of organisms from bacteria to man suggests an essential function(s) for the hs-proteins. Empirically, one function is to provide thermal protection or thermotolerance to otherwise non-permissive hs temperature (Schlesinger, M. et al. (1982) Heat shock from bacteria to man. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. p.329). Apparently those hs-proteins which are synthesized at a permissive heat shock temperature allow organisms to continue the synthesis of hs-proteins and hs-mRNA's at still higher temperatures and to survive what would be normally lethal temperatures (Key. J. L. et al. (1982) In: Heat Shock from Bacteria to Man. M. J. Schlesinger, M. Ashburner and A. Tissieres, eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. p.329). A permissive heat shock temperature is defined here as a temperature which is high enough to induce the heat shock response but not high enough to be lethal. Temperatures above break point temperature are lethal for plants which have not acquired a thermotolerance. In soybean the break point temperature is about 40.degree. C. It has previously been shown that soybean seedlings survive incubation at a lethal temperature by prior incubation at a permissive hs-temperature (Key, J. L. et al. (1983) In: NATO Advanced Studies Workshop on Genome Organization and Expression in Plants. L. Dure, ed. Plenum Press).
Several different treatment regimes of permissive heat shock result in the development of thermotolerance in the soybean seedling. These treatments include: (a) a 1- to 2-hour continuous heat shock at 40.degree. C. followed by a 45.degree. C. incubation; (b) a 30-minute heat shock at 40.degree. C. followed by 2 to 3 hours at 28.degree. C. prior to the shift to 45.degree. C.; (c) a 10-minute heat shock at 45.degree. C. followed by about 2 hours at 28.degree. C. prior to the shift to 45.degree. C.; and (d) treatment of seedlings with 50 .rho.M arsenite at 28.degree. C. for 3 hours or more prior to the shift to 45.degree. C. The important feature which these treatments have in common is the induction of synthesis and accumulation of heat shock proteins prior to incubation at the potentially lethal temperature. In fact, it has been shown that both hs-mRNA and hs-protein synthesis do occur at 45.degree. C. if the seedlings had earlier been exposed to one of the conditions described above. A likely role(s) for the hs-proteins is to protect vital functions and structures (e.g., transcription, translation and the machinery of energy production) during heat shock and to permit normal functions to return rapidly when favorable temperatures are re-established. It is known that recovery of normal mRNA and protein synthesis occurs rapidly when the temperature is shifted back to normal (e.g., 28.degree. C.) (Key, J. L. et al. (1981) Proc. Nat. Acad. Sci. USA 78:3526-3530; Schlesinger, M. J. et al. (1982) Trends Blochem. Sci. 1:222-225). The resumption of normal protein synthesis utilizes mRNA's conserved during heat shock as well as that newly synthesized during recovery, and there is no detectable synthesis of heat-shock proteins after 3-4 hours at the normal temperature. However, those heat shock proteins that were synthesized during the 40.degree. C. heat shock (recognized by the incorporation of .sup.3 H-leucine) are very stable during a subsequent chase in non-radioactive leucine, regardless of whether the chase is accomplished at 28.degree. C. or 40.degree. C.; approximately 80% of the label is retained in the heat shock proteins during a 20-hour chase.
The acquisition of thermotolerance appears to depend not only upon the synthesis of heat shock proteins but also on their selective cellular localization. In soybean seedlings, several hs-proteins become selectively localized in or associated with nuclei, mitochondria and ribosomes in a state that causes them to isolate in gradient-purified fractions of these organelles. Specifically, the complex group of 15-18 kilodalton hs-proteins selectively localize in these fractions during heat shock of soybean seedlings. The selective localization of hs-proteins is temperature dependent. The hs-proteins (except the 22-24 kd. hs-proteins which attach to the mitochondrial fraction) chase from the organelle fractions during a 4-hour incubation at 28.degree. C. and they remain organelle associated during a chase at heat shock temperature. In addition, a second heat shock following a 4-hour 28.degree. C. chase results in rapid (within 15 minutes) reassociation of hs-proteins with the organelle fractions. This association of heat shock proteins with nuclei could be explained by the hs-proteins becoming "chromatin proteins" or possibly a part of the matrix structure; both suggestions have been offered following localization studies in the Drosophila system (Arrigo, A. P. et al. (1980) Dev. Biol. 78:86-103). These findings are in basic agreement with autoradiographic results which localized hs-proteins to interband regions of polytene chromosomes (Velazquez, J. et al. (1980) Cell 20:679-689 and (1984) Cell 36:655-662).
Most of the heat shock work in plants has been done with etiolated seedlings, largely due to ease of manipulation. Heat shock proteins have not been extensively analysed in the green tissues of normal plants, but it has been shown that hs-mRNA's accumulate in green leaf tissue to levels similar to those of etiolated seedlings. Additionally, most experimental work has been done using a large temperature shift of about 10.degree. C. The response to such a non-physiological shift, however, is mimicked both at the level of hs-mRNA and hs-protein synthesis and accumulation, by a gradual increase from 28.degree. C. to 47.5.degree. C. in the case of soybean. Thus, the results from what may appear to be non-physiological experiments can be duplicated with etiolated seedlings and green plants under more normal physiological conditions of heat shock, which indeed probably occur in the normal plant environment.